Nanoparticulate compositions and formulations of piperazine compounds

ABSTRACT

The present invention relates to storage stable nanoparticulate compositions of piperazine compounds. The pharmaceutical compositions comprising the nanoparticulate compositions that are useful for the treatment and prevention of proliferative diseases including cancer are also described.

FIELD OF THE INVENTION

The present invention relates to nanoparticulate compositions in whichthe active agent is a piperazine compound and pharmaceuticalcompositions including the nanoparticulate compositions. Moreparticularly, the invention is a nanoparticulate composition thatincludes1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine.The compositions and formulations are useful for the treatment andprevention of proliferative diseases including cancer.

BACKGROUND OF THE INVENTION

1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazineand related compounds and derivatives are described in U.S. Pat. No.8,314,100, incorporated herein by reference in its entirety. Suchcompounds have been shown to have significant anti-tumor activity, buthave very poor solubility in water.

Methods of making nanoparticulate compositions are described, forexample, in U.S. Pat. Nos. 5,518,187 and 5,862,999, both for “Method ofGrinding Pharmaceutical Substances”; U.S. Pat. No. 5,718,388; for“Continuous Method of Grinding Pharmaceutical Substances”; and U.S. Pat.No. 5,510,118 for “Process of Preparing Therapeutic CompositionsContaining Nanoparticles”.

SUMMARY OF THE INVENTION

The present invention relates to nanoparticulate compositions ofpiperazine compounds such as1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazineor related compounds and derivatives as described elsewhere herein, asthe active agent, and at least one surface stabilizer.

The present invention also relates to methods of making thenanoparticulate compositions of the present invention. Such methodsinclude reducing the size of particles of a piperazine compound for atime and under conditions sufficient to provide a nanoparticulatecomposition and contacting the compound with at least one surfacestabilizer. The one or more surface stabilizers can be contacted withthe piperazine either before, during, or after size reduction ofpiperazine particles.

The present invention also relates to pharmaceutical compositions of thenanoparticulate compositions of the present invention and apharmaceutically acceptable carrier, as well as any pharmaceuticalacceptable excipients.

The present invention also relates to methods of treatment using thepharmaceutical compositions of the present invention for conditions,such as proliferative diseases or diseases that are associated with ortriggered by persistent angiogenesis.

In particular embodiments, the invention is a stable composition thatincludes:

(a) nanoparticles of compound of formula (1),

or pharmaceutically acceptable salts thereof,

wherein

-   -   X and Y are independently N or C—R⁷;    -   for the combination of variables R¹ and R²:        -   R¹ is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or halogen and R²            is F; or        -   R¹ is F and R² is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or            halogen;    -   R³ is C₁-C₃ alkyl; and    -   R⁴, R⁵, R⁶ and R⁷ are independently H, C₁-C₆ alkoxy, C₁-C₆        alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkylcarbonyl, cyano, nitro or        halogen; and

(b) at least one surface stabilizer, wherein the nanoparticles have aneffective median particle size (D50) of less than about 1,000 nm. Thecompound of formula (1) can be1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine.The at least one surface stabilizer can be at least one polyalkyleneoxide such as a block copolymer of polyethylene oxide and polypropyleneoxide. In some embodiments, the at least one surface stabilizer is apoloxamer, such as poloxamer 407 or poloxamer 338. In some embodiments,the composition is in the form of a liquid suspension. In otherembodiments, the composition is in the form of a dry solid. In someembodiments, the composition is stable after storage for at least fourweeks. In other embodiments, particularly those in the form of a powder,the composition is stable after storage for at least 6 months. In someembodiments, the effective median particle size is less than about 500nm. The ratio (wt/wt) of the compound of formula (1) to surfacestabilizer in the composition can be from about 100:1 to about 5:1.

In another aspect, the invention is a method of making a composition asrecited above by preparing a mixture of particles of a compound offormula (1), or pharmaceutically acceptable salts thereof, for example,1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine,and (b) at least one surface stabilizer, and reducing the size of theparticles of a compound of formula (1) under conditions sufficient toprovide a nanoparticulate suspension having an effective median particlesize of less than about 1,000 nm, to form a stable composition. Themethod can further include the step of drying the nanoparticulatesuspension, for example by lyophilizing a frozen suspension, to form apowder. The method can also include water in the mixture. The mixturecan include from about 5% to about 50% of the compound of formula (1)and from about 0.1% to about 5% of the surface stabilizer or, typicallyin solid forms, from about 75% to about 90% of the compound formula (1)and from about 10% to about 25% of the surface stabilizer. The ratio ofthe compound of formula (1) to surface stabilizer can be from about100:1 to about 5:1. Reducing the size of particles can be accomplishedby milling, homogenizing or precipitation, for example by wet milling.The milling can be any amount of time suitable to provide the desiredsize, for example, for about 600 minutes or about 360 minutes or anytime period in between. The suspension can be diluted with a solventsuch as water after reducing the size of the particles. A poloxamersolution can also be added to the mixture after reducing the size of theparticles.

In another aspect, the invention is a pharmaceutical composition thatincludes the nanoparticle composition as described above and at leastone pharmaceutically acceptable excipient. The at least onepharmaceutically acceptable excipient can be, for example, water orhydroxypropyl methylcellulose. The composition can be in an oral dosageform or a parenteral dosage form, and can include, for example, fromabout 0.01 to about 250 mg of the compound of formula (1), and caninclude, for example from about 0.001% to about 99.5% of the compound offormula (1).

The invention also includes a method of treating a tumor byadministering to an animal in need thereof a composition orpharmaceutical composition as described above.

Further objectives and advantages, as well as the structure and functionof preferred embodiments will become apparent from a consideration ofthe description, drawings, and examples.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1:1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazineat 400× Magnification.

FIG. 2:1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazineat 400× magnification, polarized light.

FIG. 3: Thermogravimetric analysis of1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine.

FIG. 4: Differential Scanning calorimetry Thermograph of1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention are discussed in detail below. Indescribing the embodiments, specific terminology is employed for thesake of clarity. However, the invention is not intended to be limited tothe specific terminology so selected. While specific exemplaryembodiments are discussed, it should be understood that this is done forillustration purposes only. A person skilled in the relevant art willrecognize that other components and configurations can be used withoutparting from the spirit and scope of the invention. All references citedherein are incorporated by reference as if each had been individuallyincorporated.

Piperazine compounds of formula (1),

wherein

X and Y are independently N or C—R⁷;

for the combination of variables R¹ and R²:

-   -   R¹ is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or halogen and R² is        F; or    -   R¹ is F and R² is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or        halogen;

R³ is C₁-C₃ alkyl; and

R⁴, R⁵, R⁶ and R⁷ are independently H, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆haloalkyl, C₁-C₆ alkylcarbonyl, cyano, nitro or halogen;

have been shown to be useful for the treatment of hyperproliferativedisorders. For example, such compounds can be used for treating tumors,particularly a tumor (or cancer and/or any metastases). Tumors treatablewith compounds of formula (1) include a tumor which is a breast cancer,lung cancer, gastrointestinal cancer, including esophageal, gastric,small bowel, large bowel and rectal cancer, glioma, sarcoma, such asthose involving bone, cartilage, soft tissue, muscle, blood and lymphvessels, ovarian cancer, myeloma, lymphoma, leukemia, cervical cancer,endometrial cancer, head and neck cancer, mesothelioma, renal cancer,ureter, bladder and urethral cancers, colon cancer, colorectal cancer,liver cancer, pancreatic cancer, prostate cancer, skin cancers andmelanoma. Effective treatment with a compound of formula (1) requires acomposition that can deliver the compound to the tumor. Compounds of theinvention have been tested in vitro against cancer cells lines and haveshown activities in the inhibition of cancer cell growth. For example,U.S. Pat. No. 8,314,100 describes that compounds of the invention haveshown activity against: Human OVCAR-3 (ovary), MCF-7 (breast,hormone-dependent), MDA-MB-231 (breast), PC3 (prostate), HepG2 (liver),A549 (lung), Caki-1 (kidney), HT-29 (colon), HCT116 (colon) and PANC-1(pancreas) from the American Type Culture Collection (ATCC) (Manassas,Va.); MKN-45 (stomach) from DSMZ (Germany); UMRC2 (kidney) from the U.S.National Cancer Institute (Bethesda, Md.); Huvec (human umbilical veinendothelial cells), HEK293 (human embryonic kidney) and SK-OV-3 (overy)from Korean Cell Line Bank (Seoul, Korea).

An example of a compound of formula (1) is1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine(Compound A):

Compound A has very poor solubility in water, less than 0.1 μg/ml. Thispoor solubility in water makes it difficult to deliver the compound andrelated compounds of formula (1) and has limited clinical development,thus creating a need for the development of a formulation suitable forclinical use. The present invention was developed with a goal ofobtaining a suitable formulation that can provide a stable suspension ofthe compound at a concentration of about 100 mg/mL or a driedformulation that could be either delivered orally or used to prepare astable suspension. While the invention is described and exemplifiedprimarily with respect to1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine,it is to be understood that the invention is applicable to othercompounds of formula (1).

In order to improve bioavailability, the present formulation wasdeveloped with the concept of obtaining a submicron particle sizedistribution of the piperazine compound in a suspension using a minimumof excipients that are generally regarded as safe (GRAS) to reducepotential toxic interference during the study. Test articles of theprototype formulation will be made from a scaled-up process underbest-clean conditions. Additionally, the remaining suspension isintended to serve as the basis for future work in developing asolid-dosage formulation of, for example,1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine.

“Active agent”, “drug”, “Active Pharmaceutical Ingredient”, or “API” asused herein, refers to compounds of formula (1),

wherein

X and Y are independently N or C—R⁷;

for the combination of variables R¹ and R²:

R¹ is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or halogen and R² is F; or

R¹ is F and R² is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or halogen;

R³ is C₁-C₃ alkyl; and

R⁴, R⁵, R⁶ and R⁷ are independently H, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆haloalkyl, C₁-C₆ alkylcarbonyl, cyano, nitro or halogen. Exemplaryembodiments of compounds of formula (1) include the following:

Compound R¹ R² R³ R⁴ R⁵ R⁶ X Y Compound A F H Me OMe H OMe C—H C—HCompound B F H Me Me H Me C—H C—H Compound C F H Me H H H C—OMe C—HCompound D F H Me OMe H H C—H C—H Compound E F H Me Me H H C—H C—HCompound F F H Me Cl H H C—H C—HThe active agent can be in a free form, or pharmaceutically acceptablesalt form, in the form of their possible enantiomers, diasteromers andrelative mixtures, polymorphs, amorphous, partially amorphous forms,solvates (including hydrates), their active metabolites and prodrugs.

“Poorly water soluble”, as used herein, has the meaning generallyattributable in the art. For example, poorly water soluble can meanhaving a solubility in water at 20° C. of less than 1%, e.g., 0.01%weigh/volume, i.e., a “sparingly soluble to very slightly soluble drug”as described in Remington: The Science and Practice of Pharmacy, 19thEdition, A. R. Gennaro, Ed., Mack Publishing Company, US, Vol. 1, p. 195(1995). Other similar generally recognized definitions are encompassedby the term.

By “an effective median particle size of less than about 1,000 nm” it ismeant that at least 50% of the nanoparticulate active agent particleshave a particle size of less than about 1,000 nm, as determined on thebasis of the weight average particle size as measured by conventionalparticle size measuring techniques well-known to those skilled in theart. Such techniques include light scattering methods, microscopy andother conventional techniques, e.g., sedimentation field flowfractionation, photon correlation spectroscopy, light scattering anddisk centrifugation. As will be recognized, similar language related toother effective particle sizes have similar definitions. Mean or averageparticle size can be determined similarly. Designations of particlesizes and other description of particle size will be recognized bypersons skilled in the art. For example, D50, the median, describes acomposition with a population of nanoparticulate active agent particleswhere half of the population has a diameter below this value. Similarly,D90 describes the population where the diameter of ninety percent of thedistribution has a smaller particle size and ten percent has a largerparticle size. The D10 describes the population where the diameter often percent of the particles is smaller than and ninety percent of theparticles larger than the stated value.

The terms “effective amount” or “pharmaceutically effective amount” of ananoparticle formulation or composition, as provided herein, refer to anontoxic but sufficient amount of the nanoparticle formulation orcomposition to provide the desired response and correspondingtherapeutic effect, in an amount sufficient to effect treatment of thesubject. As will be pointed out below, the exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the condition being treated,mode of administration and the like. An appropriate “effective” amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation.

The phrase “pharmaceutically acceptable” or “pharmacologicallyacceptable” means a material which is not biologically or otherwiseundesirable, i.e., the material may be administered to an individualalong with the nanoparticle formulation or composition without causingany undesirable biological effects or interacting in a deleteriousmanner with any of the components of the composition in which it iscontained.

The active pharmaceutical ingredient (API),1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine,as well as related compounds described herein, is being investigated asan anti-cancer treatment for solid tumors that has the potential to bewell absorbed by the intestine when tested in vitro and thatdemonstrates good oral bioavailability in animal model studies. Becauseof the poor aqueous solubility of1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine,production of a submicron suspension needed to be developed as a viableoral delivery option to avoid the need to administer large volumes of a1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazinesolution to achieve the intended dosage levels.

In exemplary embodiments, the invention is a formulation that is astable nanoparticulate composition of a compound of formula (1), forexample1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine(Compound A) having an effective median particle size (D50) of less thanabout 1,000 nm and preferably at least one surface stabilizer. Thecomposition can be in the form of a suspension, typically in water, or adried powder. In exemplary embodiments of the invention in dried form,the nanoparticulate composition can be redispersed such that theeffective median particle size of the redispersed compound of formula(1) is less than about 1,000 nm. This is significant in that it allowsthe nanoparticulate compound to redisperse to a substantially smallparticle size to provide benefits similar to those of the suspensionhaving a similar nanoparticulate particle size.

In exemplary embodiments, the particles in a suspension, whether asoriginally prepared, as a redispersed solid, or in a solid form, canhave an effective median particle size of less than about 1,000 nm, lessthan 500 nm, less than about 250 nm, or less than about 100 nm. Inexemplary embodiments, the particles in a suspension, whether anoriginally prepared or as a redispersed solid, or in a solid form canhave an effective mean particle size of less than about 1,000 nm, lessthan 500 nm, less than about 250 nm, or less than about 100 nm. Inexemplary embodiments, the median or mean particle size can be, forexample, from about 100 nm to about 1,000 nm, from about 100 nm to about500 nm, from about 200 nm to about 1,000 nm, from about 200 nm to about500 nm, from about 100 nm to about 200 nm, or from about 250 nm to about500 nm. In exemplary embodiments, the particles in a suspension, whetheras originally prepared, as a redispersed solid, or in a solid form canhave an effective median or mean particle size of more than about 100nm, more than about 250 nm, or more than about 500 nm.

In accordance with the present invention, the active agent may bepresent in an amount by weight of from about 0.001% to about 99.5%, fromabout 0.1% to about 95% by the dried weight of the composition orformulation. In some embodiments, the active agent is present in anamount of from about 0.5% to about 90% by the dried weight of thecomposition or formulation. In some embodiments, the active agent may bepresent in an amount of from about 5% to about 50%, for example fromabout 5% to about 40%, from about 10% to about 30%, or from about 10% toabout 40%. In some embodiments of the invention that are in the form ofa suspension, the active agent may be present in an amount of from about5% to about 50%, for example from about 5% to about 40%, from about 10%to, about 30%, or from about 10% to about 40%. In other exemplaryembodiments, the active agent may be present in an amount of from about75% to about 99%, for example from about 75% to about 90%, from about80% to about 99%, or from about 90% to about 99%. In other exemplaryembodiments of a dried form of the composition, the active agent may bepresent in an amount of from about 75% to about 99%, for example fromabout 75% to about 90%, from about 80% to about 99%, or from about 90%to about 99%. In other exemplary embodiments of a dried form of thecomposition, the active agent may be present in an amount of about 75%,about 80%, about 90%, about 95%, or about 99%.

Methods of characterizing the API included not only optical methods, butalso thermal methods, as all milling processes, including those ofrelatively low-energy, can generate localized heating at the particulatelevel. Thermal data can be used to determine if the active agentexhibits any thermal transitions that could be detrimental to particlesize reduction under milling stresses.

As the simplest and most practical size reduction method, media millingwas chosen to initiate the study. In media milling, the API is mixedwith a milling aid, typically a surfactant, and made into slurry withmilling media, spherical beads of a hard, inert material. Particles arebroken down through mechanical abrasion by agitating the slurry, eitherby low-energy means, such as rolling it in a container on a roller mill,or by high-energy means, such as mixing it with a rotary agitator, knownas a spindle mill.

Several GRAS milling aids were selected for their suitability in oraldosage forms, and were tested for milling efficacy at differentconcentrations with different levels of API. From these developmentsamples, potential candidates were selected and then monitored forparticle-size stability over time. The leading candidate from these wasscaled-up and used to process API in bulk for toxicity studies under GLPbest-clean conditions.

Other methods of size reduction, such as microfluidization andnucleation or precipitation, were considered as alternatives had mediamilling proved unsuccessful and can be used in embodiments of theinvention. Microfluidization involves forcing a suspension of the APIthrough a narrow aperture under high pressure (up to 25,000 psi) usingthe shear forces so generated to break apart the particles. To reduceparticles in size by precipitation, the API is dissolved in a suitablesolvent and then combined with a miscible antisolvent; whichdestabilizes the solution, and causes the API to precipitate. Variationsin the rate of solvent/antisolvent combination as well as the additionof segregating agents and high-energy disruption can producereduced-size particles.

In the present invention, roller milling was chosen to process thecompound of formula (1) (Compound A as an example) because it is anuncomplicated procedure for particle size reduction that not onlyresults in high process yields, even in small test batches, but alsoposes a minimal likelihood of personnel exposure to highly potent API.Sterile Water for Injection (SWFI) was used instead of purified water toreduce the potential for microbial contamination.

Surface Stabilizer

Surface stabilizers which can be employed in the invention include, butare not limited to, known organic and inorganic pharmaceuticalexcipients. Such excipients include various polymers, low molecularweight oligomers, natural products and surfactants. Surface stabilizersinclude nonionic type, cationic type, anionic type, and zwitterionictype surfactants. Examples of nonionic surface stabilizers includeethoxylated aliphatic alcohol, polyoxyethylene surfactants, carboxylicesters, polyethylene glycol esters, anhydrosorbitol ester and itsethoxylated derivatives, glycol esters of fatty acids, carboxylicamides, monoalkanolamine condensates, and polyoxyethylene fatty acidamides. Examples of cationic surface stabilizers include quaternaryammonium salts, amines with amide linkages, polyoxyethylene alkyl andalicyclic amines, N,N,N′,N′ tetrakis substituted ethylenediamines, and2-alkyl 1-hydroxyethyl 2-imidazolines. Examples of anionic surfacestabilizers include carboxylates, sulphonates, petroleum sulphonates,alkylbenzenesulphonates, naphthalenesulphonates, olefin sulphonates,alkyl sulphates, sulphates, sulphated natural oils and fats, sulphatedesters, sulphated alkanolamides and alkylphenols, ethoxylated andsulphated. Examples of zwitterionic surface stabilizers include N-coco3-aminopropionic acid/sodium salt, N-tallow 3-iminodipropionate,disodium salt, N-carboxymethyl-N-dimethyl-N-9 octadecenyl ammoniumhydroxide and N-cocoamidethyl-N-hydroxyethylglycine, sodium salt.

Representative examples of surface stabilizers include poloxamers, whichare block copolymers of ethylene oxide and propylene oxide, gelatin,casein, lecithin (phosphatides), dextran, gum acacia, cholesterol,tragacanth, stearic acid, benzalkonium chloride, calcium stearate,glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifyingwax, sorbitan esters, polyoxyethylene alkyl ethers (e.g., macrogolethers, such as cetomacrogol 1000), polyoxyethylene castor oilderivatives, polyoxyethylene sorbitan fatty acid esters (e.g., thecommercially-available Tweens®, such as, e.g., Tween 20® and Tween® (ICISpecialty Chemicals)); polyethylene glycols (e.g., Carbowax 3550® andCarbowax 934® (Union Carbide)), polyoxyethylene stearates, colloidalsilicon dioxide, phosphates, carboxymethylcellulose calcium,carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose phthalate, noncrystalline cellulose,magnesium aluminium silicate, triethanolamine, polyvinyl alcohol (PVA),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol, superione and triton); andpoloxamines.

Exemplary surface stabilizers include Pluronics®, e.g., Poloxamer 105(Pluronic® L35), Poloxamer 108 (Pluronic® F38), Poloxamer 124 (Pluronic®L44NF), Poloxamer 184 (Pluronic® L-64), Poloxamer 188 (Pluronic® F68NF),Poloxamer 237 (Pluronic® F87NF), Poloxamer 238 (Pluronic® F88),poloxamer 338 (Pluronic® F108NF), Poloxamer 401 (Pluronic® L121),Poloxamer 407 (Pluronic®F127NF) and other poloxamer products;Tetronics®, e.g., Tetronic 904, 908, 1107, and 90R4, which aretetrafunctional block copolymers derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine (BASF WyandotteCorporation, Parsippany, N.J.)); Tetronic 15080 (T-1508) (BASF WyandotteCorporation), Tritons X-2000, which is an alkyl aryl polyether sulfonate(Rohm and Haas); Crodestas F-100®, which is a mixture of sucrosestearate and sucrose distearate (Croda Inc.);p-isononylphenoxypoly-(glycidol), also known as Olin-10G® or Surfactant10-Go (Olin Chemicals, Stamford, Conn.); Crodestas SL-400 (Croda, Inc.);and SA9OHCO, which is C18H37CH2(CON(CH3)-CH2(CHOH)4(CH2OH)2 (EastmanKodak Co.); decanoyl-N-methylglucamide; n-decyl-beta-D-glucopyranoside;n-decyl-beta-D-maltopyranoside; n-dodecyl-beta-D-glucopyranoside;n-dodecyl-beta-D-maltoside; heptanoyl-N-methylglucamide;n-heptyl-beta-D-glucopyranoside; n-heptyl-beta-D-thioglucoside;n-hexyl-beta-D-glucopyranoside; nonanoyl-N-methylglucamide;n-nonyl-beta-D-glucopyranoside; octanoyl-N-methylglucamide;n-octyl-beta-D-glucopyranoside; octyl-beta-D-thioglucopyranoside;PEG-phospholipid, PEG-cholesterol, PEG-cholesterol derivative,PEG-vitamin A, PEG-vitamin E, lysozyme, random copolymers of vinylpyrrolidone and vinyl acetate and the like.

Examples of useful cationic surface stabilizers include, but are notlimited to, polymers, biopolymers, polysaccharides, cellulosics,alginates, phospholipids, and nonpolymeric compounds, such aszwitterionic stabilizers, poly-n-methylpyridinium, anthryl pyridiniumchloride, cationic phospholipids, chitosan, polylysine,polyvinylimidazole, polybrene, polymethylmethacrylate trimethylammoniumbromide (PMMTMABr), hexyldesyltrimethylammonium bromide (HDMAB), andpolyvinylpyrrolidone-2-dimethylaminoethyl methacrylate dimethyl sulfate.Such exemplary cationic surface stabilizers and other useful cationicsurface stabilizers are described in J. Cross and E. Singer, CationicSurfactants: Analytical and Biological Evaluation, Marcel Dekker (1994);P. and D. Rubingh, Ed., Cationic Surfactants: Physical Chemistry, MarcelDekker (1991); and J. Richmond, Cationic Surfactants: Organic Chemistry,Marcel Dekker (1990).

Most of these surface stabilizers are known pharmaceutical excipientsand are described in detail in the Handbook of PharmaceuticalExcipients, published jointly by the American Pharmaceutical Associationand The Pharmaceutical Society of Great Britain (The PharmaceuticalPress, 2000), specifically incorporated by reference. The surfacestabilizers are commercially available and/or can be prepared bytechniques known in the art.

Combinations of more than one surface stabilizer can be used in theinvention. Exemplary primary surface stabilizers include, but are notlimited to, poloxamers, hydroxypropyl methylcellulose,hydroxypropylcellulose, polyvinylpyrrolidone, random copolymers of vinylpyrrolidone and vinyl acetate or a combination thereof. Exemplarysecondary surface stabilizers include, but are not limited to, sodiumlauryl sulfate and dioctylsulfosuccinate.

In exemplary embodiments, the at least one surface stabilizer is apoloxamer. Exemplary poloxamers useful for the invention can have amolecular weight of from about 9,000 to about 20,000. Specific exemplarypoloxamers that can be used in the invention include poloxamer 407 andpoloxamer 338 or equivalent materials such as corresponding Pluronics.

The concentration of the at least one surface stabilizer can vary fromabout 0.5% to about 99.999%, from about 5.0% to about 99.9%, from about1.0% to about 99.0%, or from about 10% to about 99.5%, by weight, basedon the total combined dry weight of the active agent and at least onesurface stabilizer, not including other excipients. If a combination oftwo or more surface stabilizers is employed in the composition, theconcentration of at least one primary surface stabilizer can vary fromabout 0.01% to about 99.5%, from about 0.1% to about 95%, or from about0.5% to about 90%, by weight, based on the total combined dry weight ofthe active agent not including other excipients.

In some embodiments, the surface stabilizer may be present in an amountof from about 0.1% to about 5%, for example from about 0.1% to about2.5%, from about 0.1% to about 1%, or from about 0.25% to about 1%. Insome embodiments of the invention that are in the form of a suspension,the surface stabilizer may be present in an amount of from about 0.1% toabout 5%, for example from about 0.1% to about 2.5%, from about 0.1% toabout 1%, or from about 0.25% to about 1%. In some embodiments of theinvention that are in the form of a suspension, the surface stabilizermay be present in an amount of about 0.1%, about 0.025%, about 1%, about2%, or about 2.5%. In other exemplary embodiments, the surfacestabilizer may be present in an amount of from about 1% to about 20%,for example from about 10% to about 25%, from about 1% to about 20%, orfrom about 1% to about 10%. In other exemplary embodiments of a driedform of the composition, the surface stabilizer may be present in anamount of from about 1% to about 20%, for example from about 10% toabout 25%, from about 1% to about 20%, or from about 1% to about 10%. Inother exemplary embodiments of a dried form of the composition, thesurface stabilizer may be present in an amount of about 25%, about 20%,about 10%, about 5%, or about 1%.

Embodiments of the invention can include a ratio of active agent tosurface stabilizer in the range of from about 100:1 to about 5:1. Insome embodiments, the ratio of active agent to surface stabilizer isfrom about 200:1 to about 1:1, from about 100:1 to about 10:1, fromabout 20:1 to about 5:1, or from about 15:1 to about 10:1. In exemplaryembodiments, which the ratio of active agent to surface stabilizer isabout 100:1, about 50:1, about 25:1, about 10:1, about 12.5:1, about5:1.

Processes for Preparing the Nanoparticle Compositions

The nanoparticulate compositions of the present invention can be madeusing, e.g., milling, homogenization or precipitation techniques.

API morphology can be characterized by optical microscopy (e.g., OlympusBX51 microscope with Clemex JS-2000 controller). Differential scanningcalorimetry (e.g., Mettler-Toledo DSC 1) and thermogravimetric analysis(e.g., Mettler-Toledo TGA/DSC 1) can be used to measure the thermalcharacteristics of the material. Particle size measurements can be madethroughout by, for example, laser diffraction (e.g. Horiba LA-950V2), bydispersing the material, either in water, when surfactant is present inthe test sample or in a dilute solution of poloxamer when no otherdispersant was present.

Prototype formulations are processed in glass sample vials on a rollermill (U.S. Stoneware) using a slurry of 0.5 mm diameteryttria-stabilized zirconia ceramic milling media (from, e.g. Tosoh). GLPmilling is done in 2 L media bottles of Type 1 borosilicate glass.Preparations of the test article material are done under “best-clean”conditions, under which all contact materials and equipment aresanitized either by application of 70% isopropanol or by autoclaving.All such preparations are performed using aseptic technique in asanitized laminar flow hood (Airclean 600).

Milling the active agent to obtain a nanoparticulate dispersion involvesdispersing particles of the active agent in a liquid dispersion mediumin which the active agent is poorly soluble, followed by applyingmechanical means in the presence of milling media to reduce the particlesize of the active agent to the desired effective average particle size.The dispersion medium can be, e.g., water, ethanol, t-butanol, glycerin,polyethylene glycol (PEG), hexane or glycol.

In embodiments, aqueous nanomilling of the active agent is conducted inthe presence of a hydrophilic stabilizer of the surface stabilizer. Forexample, the active agent particles can be reduced in size in thepresence of the at least one surface stabilizer. Alternatively, theactive agent particles can be contacted with one or more surfacestabilizers after attrition. Other compounds, such as a diluent, can beadded to the active agent/surface stabilizer composition either before,during or after the size reduction process. Dispersions can bemanufactured continuously or in a batch mode.

In other embodiments, the nanoparticulate composition is prepared bymicroprecipitation. This is a method of preparing stable dispersions ofpoorly soluble active agents in the presence of one or more surfacestabilizers and one or more colloidal stability enhancing surface activeagents free of any trace toxic solvents or solubilized heavy metalimpurities. Such a method comprises, e.g., (1) dissolving the activeagent in a suitable solvent; (2) adding the formulation from step (1) toa solution comprising at least one surface stabilizer; and (3)precipitating the formulation from step (2) using an appropriatenon-solvent or anti-solvent. The method can be followed by removal ofany formed salt, if present, by dialysis or diafiltration andconcentration of the dispersion by conventional means.

In yet other embodiments, the nanoparticle compositions are prepared byhomogenization methods. Such methods include the step of dispersing theactive agent particles in a liquid dispersion medium, followed bysubjecting the dispersion to homogenization to reduce the particle sizeof the active agent to the desired effective average particle size. Theactive agent particles can be reduced in size in the presence of atleast one surface stabilizer. Alternatively, the active agent particlescan be contacted with one or more surface stabilizers either before orafter attrition. Other compounds, such as a diluent, can be added to theactive agent/surface stabilizer composition either before, during, orafter the size reduction process. Dispersions can be manufacturedcontinuously or in a batch mode.

Compositions according to the invention can be prepared in either theform of a suspension or as a dry powder. For preparation of asuspension, the active agent is reduced in size using one of thepreviously described techniques. The size reduction may be accomplishedusing the active agent alone, the active agent dispersed in a solvent,for example water or another solvent as mentioned above, the activeagent in combination with the surface stabilizer or a combination of theactive agent, solvent and surface stabilizer. Size reduction iscontinued until the desired particle size of the active agent isachieved. Additional surface stabilizer can be added to achieve thefinal desired concentration. Furthermore, if desired, the suspension canbe diluted with a suitable solvent to reach a desired concentration ofactive agent. If a solvent is present during size reduction, dilutioncan be accomplished by adding additional solvent, which may the same ordifferent than the solvent used during size reduction. The term solventas used herein includes a single solvent or a mixture of solvents. Asdescribed above, additional components may be present during the sizereduction process or may be added afterward as desired.

In an exemplary embodiment, a suspension is prepared by dissolving thesurface active agent, for example a poloxamer such as poloxamer 407 orpoloxamer 338, in approximately ⅓ of the final amount of solvent, forexample purified water, in a suitable container. The active agent, forexample,1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine,is suspended in the solution of surface active agent. Milling media isadded to the container. The suspension is subjected to size reductionuntil the desired particle size is achieved. For example, the suspensionis milled until the D50 or D90 of the particle-size distribution,measured by, for example, laser diffraction, is below a 1,000 nm. Thesuspension is then removed from the milling media. The media is rinsedand the suspension diluted with solvent to achieve the desired finalconcentration of active agent, for example 10%. After preparation, thesuspension can be assayed to confirm that the particle size andconcentration is at the desired level.

Dry formulations can be obtained by removing solvent from thesuspension. Solvent removal can be conducted on either the suspension asobtained immediately after size reduction or after further dilution. Anysuitable method of drying may be used that results in a stableformulation. Exemplary methods of drying include spray drying,supercritical drying, drum drying, dielectric drying, natural airdrying, Refractance Window™ drying, Infrared Zone Drying™ and freezedrying (lyophilization). In an exemplary method of drying, thesuspension is freeze dried by being pre-frozen using liquid nitrogen andlyophilized in bulk on pre-chilled shelves to produce a dry powder.

Stability

Formulations and compositions according to the present invention arestable, and in particular are stable upon storage. In the presentcontext, a stable formulation or composition is a formulation orcomposition in which the active agent does not degrade or decompose uponstorage and in which the particle size distribution does not changesignificantly. The particle size does not change significantly if, uponstorage, the median particle size does not increase to greater than1,000 nm. In exemplary embodiments, the median particle size does notincrease to greater than 900 nm, greater than 800 nm, greater than 700nm, greater than 600 nm, or greater than 500 nm, upon storage. As usedherein, the active agent does not degrade or decompose upon storage if,after storage, at least 95% of the originally present active agentremains. In exemplary embodiments, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% of the original amount of activeagent is present after storage.

Storage conditions for stability testing can be varied and testing maybe accomplished under typical storage conditions or under acceleratedstorage conditions. As used herein, stable under storage conditions or“storage stable” means that the composition is stable when stored at 5°C. for one week. Some embodiments of the invention are storage stablefor even longer periods of time, for example after storage at 5° C. fortwo weeks, for three weeks, or for four weeks. Capsules containing thedried formulation can be stable for even longer periods, for example upto one month, up to two months, up to three months, up to four months,up to five months, up to six two months, or even longer.

Pharmaceutical Compositions and Methods of Treatment

The pharmaceutical compositions of the present invention also includeone or more excipients. Excipients include physiologically acceptablecarriers, adjuvants or vehicles, collectively referred to as carriers.The compositions can be formulated for oral administration in solid, orliquid form, and the like.

Excipients can include one or more binding agents, filling agents,lubricating agents, suspending agents, sweeteners, flavoring agents,preservatives, buffers, wetting agents, disintegrants, effervescentagents and other excipients. Such excipients are known in the art.Examples of filling agents include lactose monohydrate, lactoseanhydrous, microcrystalline cellulose, such as Avicel® PH101 and Avicel®PH102, microcrystalline cellulose and silicified microcrystallinecellulose (ProSolv SMCC®), and various starches; examples of bindingagents are various celluloses and cross-linked polyvinylpyrrolidone.Suitable lubricants, including agents that act on the flowability of thepowder to be compressed, include colloidal silicon dioxide, such asAerosil® 200, talc, stearic acid, magnesium stearate, calcium stearateand silica gel. Sweeteners can be any natural or artificial sweetener,such as, for example, sucrose, xylitol, sodium saccharin, cyclamate,aspartame, sucralose, maltitol and acsulfame. Examples of flavoringagents include Magnasweet® (trademark of MAFCO), bubble gum flavor, andfruit flavors, and the like. Examples of preservatives include potassiumsorbate, methylparaben, propylparaben, benzoic acid and its salts, otheresters of parahydroxybenzoic acid, such as butylparaben; alcohols, suchas ethyl or benzyl alcohol. Suitable diluents include pharmaceuticallyacceptable inert fillers, such as microcrystalline cellulose, lactose,dibasic calcium phosphate, saccharides and/or mixtures of any of theforegoing. Examples of diluents include microcrystalline cellulose, suchas Avicel® PH101 and Avicel® PH1 02; lactose, such as lactosemonohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calciumphosphate, such as Emcompress®; mannitol; starch; sorbitol; sucrose; andglucose. Suitable disintegrants include lightly crosslinked polyvinylpyrrolidone, corn starch, potato starch, maize starch, and modifiedstarches, croscarmellose sodium, cross-povidone, sodium starch glycolateand mixtures thereof. Examples of effervescent agents are effervescentcouples, such as an organic acid and a carbonate or bicarbonate.Suitable organic acids include, e.g., citric, tartaric, malic, fumaric,adipic, succinic and alginic acids and anhydrides and acid salts.Suitable carbonates and bicarbonates include, e.g., sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate,magnesium carbonate, sodium glycine carbonate, L-lysine carbonate andarginine carbonate. Alternatively, only the sodium bicarbonate componentof the effervescent couple may be present.

The nanoparticulate compositions of the invention can be administered toa subject via any conventional means including orally and parenterally.As used herein, the term “subject” is used to mean an animal, preferablya mammal, including a human or non-human. The terms patient and subjectmay be used interchangeably.

Solid dosage forms for oral administration include, but are not limitedto, capsules, tablets, pills, powders and granules. In such solid dosageforms, the present nanoparticle composition can be admixed with at leastone of the following: (a) one or more inert excipients (or carriers),such as sodium citrate or dicalcium phosphate; (b) fillers or extenders,such as starches, lactose, sucrose, glucose, mannitol and silicic acid;(c) binders, such as carboxymethylcellulose, alignates, gelatin,polyvinylpyrrolidone, sucrose and acacia; (d) humectants, such asglycerol; (e) disintegrating agents, such as cross-linked starches,polyvinylpyrrolidone XL, agar-agar, calcium carbonate, potato or tapiocastarch, alginic acid, certain complex silicates and sodium carbonate;(f) solution retarders, such as paraffin; (g) absorption accelerators,such as quaternary ammonium compounds; (h) wetting agents, such as cetylalcohol and glycerol monostearate; (i) adsorbents, such as kaolin andbentonite; and (j) lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate or mixturesthereof. For capsules, tablets and pills, the dosage forms may alsocomprise buffering agents.

Liquid nanoparticulate dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrupsand elixirs. In addition to the present nanoparticle composition, theliquid dosage forms may include excipients such as inert diluentscommonly used in the art, such as water or other solvents, co-solvents,solubilizing agents and emulsifiers. Non-limiting examples of solventsand co-solvents include ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, such ascottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, andsesame oil, glycerol, tetrahydrofurfuryl alcohol and dimethylisosorbide, polyethyleneglycols, fatty acid esters of sorbitan, ormixtures of these substances, and the like. The composition can alsoinclude adjuvants, such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring and perfuming agents.

Dosage unit compositions may contain such amounts of such submultiplesthereof as may be used to make up the daily dose. Any dosage amount maybe present in a pharmaceutical composition for oral delivery. Forexample, the solid dosage form for oral delivery may include, forexample, from about 0.1 mg to about 500 mg active agent, from about 1 mgto about 500 mg active agent, from about 10 mg to about 250 mg activeagent, or any other suitable or desired amount. The solution orsuspension form for oral and parenteral delivery may include, forexample, from about 1% to about 50% active agent, from about 5% to about30% active agent, from about 10% to about 20% active agent, or any othersuitable or desired amount in a solution or suspension. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors: the type and degree ofthe cellular or physiological response to be achieved; activity of thespecific agent or composition employed; the specific agents orcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration andrate of excretion of the agent; the duration of the treatment; drugsused in combination or coincidental with the specific agent; and likefactors well-known in the medical arts.

The pharmaceutical compositions of the present invention are useful fortreating proliferative diseases or diseases that are associated with ortriggered by persistent angiogenesis. A proliferative disease is mainlya tumor disease (or cancer) (and/or any metastases). The inventivecompositions are particularly useful for treating a tumor which is abreast cancer, lung cancer, gastrointestinal cancer, includingesophageal, gastric, small bowel, large bowel and rectal cancer, glioma,sarcoma, such as those involving bone, cartilage, soft tissue, muscle,blood and lymph vessels, ovarian cancer, myeloma, lymphoma, leukemia,female cervical cancer, endometrial cancer, head and neck cancer,mesothelioma, renal cancer, ureter, bladder and urethral cancers, coloncancer, colorectal cancer, liver cancer, pancreatic cancer, prostatecancer, skin cancers and melanoma. Compounds of the invention haveparticularly shown effectiveness with respect to ovarian, breastincluding hormone-dependent breast, prostate, liver, lung, kidney,colon, pancreatic, stomach, and endothelial cancers. The pharmaceuticalcompositions of the present invention can be combined with otherchemotherapeutics to treat a tumor or are useful for a treating a tumorthat is refractory to treatment with other chemotherapeutics due tomultidrug resistance.

The following non-limiting examples are given to illustrate the presentinvention. It should be understood, however, that the invention is notto be limited to the specific conditions or details described in theseexamples.

EXAMPLES Example 1 Classical formulation of1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine

1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazinehas a very poor solubility in water, less than 0.1 m/ml, and itssolubility was tested in several co-solvents. In most cases, the initialclear solution showed varying amounts of precipitation at differentpoints in the dilution schedule. Thus, these classical formulations arenot useful in clinical use, although they could be used in some limitedpurpose in pre-clinical studies.

Change of Formulation Concentration formulation DMAC/Cremophor EL/DIwater <5 mg/ml n/a (10/10/80 vol %) DMAC/Tween 80/DI water ~1 mg/ml n/a(10/10/80 vol %) 10% Tween 80 in 65% PEG300 3.7 mg/ml n/a 10% DMAC:90%of 35% 10 mg/ml precipitants at Solutol ® HS 15 in water bottom after 5hr 5% DMAC:95% of 50% 10 mg/ml precipitants at Solutol ® HS 15 in waterbottom after 3 hr *DMAC: N,N-Dimethylacetamide

Example 2 Preparation of Nanoformulation 2.1 Characterization of1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine

1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazinewas a freely flowing, off-white powder that was practically insoluble inwater. Particles were crystalline and irregularly shaped. FIG. 1illustrates particles of1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazineat 400× Magnification. FIG. 2 illustrates1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazineat 400× magnification under polarized light.

Based on chemical structure, the refractive index of1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazinewas predicted to be 1.637. For particle size measurements, the imaginarycomponent of the refractive index, or the i-value (a unitless factorused by the laser diffraction algorithm to account for the absorption oflight by the particles) was determined to be 0.1. Unmilled1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazinedispersed readily in an aqueous solution of (Aerosol OT). The medianparticle size (D50) measured at 13 μm.

Thermogravimetric analysis (see FIG. 3) showed a mass loss of 5.8% at anonset temperature of 105° C., consistent with loss of moisture. Massloss after 220° C. may be attributable to thermal degradation.

Differential scanning calorimetry (see FIG. 4) confirmed the endothermrelated to the initial mass loss at 108° C.1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazinemelted starting at 163° C. and showed no indication of decompositionuntil 223° C., confirming the gradual mass loss in the TGA analysis.

These data indicated that the material was thermally stable. From theseresults, no thermal phenomena were determined that might have adetrimental effect on the milling process.

2.2 Evaluation of Particle Size Reduction

The milling aids selected for the size-reduction trials were poloxamer407, poloxamer 338, sodium lauryl sulfate, and sodiumcarboxymethylcellulose. Each was tested at both 0.25% and 1%, by weight.Each of these test solutions was used to mill1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine(API) at both 12.5% and 25%, by weight. Samples were rolled and testedat intervals, depending on the observed progress of the particle sizereduction. The data are shown in Table 1. The poloxamers appeared toperform better at higher concentrations with less API, while sodiumlauryl sulfate performed better at lower concentration with more API inshort time milling (120 minutes), but with less API in longer timemilling (240 minutes and 300 minutes), and sodium carboxymethylcellulosedid not prove to be an effective milling aid for API. After 360 minutesof milling, the two most promising candidates were 1% poloxamer 407 with12.5% API and 1% poloxamer 338 with 12.5% API. All others showedattenuation in efficacy, no appreciable efficacy, or an increase inparticle size, either due to agglomeration caused by over-milling orripening. (As understood in the art, ripening occurs when small crystalsor particles dissolve and redeposit onto larger crystals or particles.)The two poloxamer preparations were maintained at both ambientconditions and at 5° C. as an informal assessment of their particle-sizestability. The measurements remained virtually unchanged over four weeksas shown in Table 2.

TABLE 1 Initial dispersant selection data Median Particle Size (D50) at:Dispersant API 0 min 120 min 240 min 300 min 360 min Poloxamer 407 0.25%12.5% 13 1.9 μm 3.8 μm 3.1 μm N/A   25% 5.3 μm 4.2 μm 6.0 μm N/A   1%12.5% 0.4 μm 1.2 μm 0.2 μm 0.1 μm   25% 1.3 μm 2.5 μm 2.1 μm N/APoloxamer 338 0.25% 12.5% 13 2.4 μm 0.5 μm 1.3 μm N/A   25% 8.4 μm 5.9μm 5.9 μm N/A   1% 12.5% 0.2 μm 0.2 μm 0.2 μm 0.2 μm   25% 6.3 μm 3.5 μm4.7 μm N/A Sodium Lauryl 0.25% 12.5% 13 1.4 μm 0.2 μm 0.9 μm N/A Sulfate  25% 0.3 μm 1.8 μm 2.4 μm N/A   1% 12.5% 3.0 μm 2.7 μm 1.2 μm N/A   25%3.5 μm 3.3 μm 2.8 μm N/A Sodium 0.25% 12.5% 13  15 μm N/A N/A N/ACarboxymethylcellulose   25%  14 μm N/A N/A N/A   1% 12.5%  12 μm N/AN/A N/A   25%  16 μm N/A N/A N/A

TABLE 2 Particle-size stability of 12.5% API trial preps Median ParticleSize (D50) at: Prep Condition Initial 1 Week 2 Weeks 3 Weeks 4 Weeks 1%Poloxamer Ambient 0.2 μm 0.2 μm 0.1 μm 0.1 μm 0.1 μm 407 5° C. 0.2 μm0.2 μm 0.1 μm 0.1 μm 1% Poloxamer Ambient 0.2 μm 0.2 μm 0.2 μm 0.2 μm0.2 μm 338 5° C. 0.2 μm 0.2 μm 0.2 μm 0.2 μm

The suspension of 25% API in 0.25% sodium lauryl sulfate (SLS) showedinitial promise under the test conditions, but resulted in eventualparticle-size increase (e.g., by ripening) during milling. In order torule out the possibility of particle fusion by overmilling, a secondpreparation was made by milling only a total of 120 minutes, and thenmonitored for particle size. After one week, the preparation showedevidence of ripening as shown in Table 3:

TABLE 3 Particle-size stability of SLS Repreparation Median ParticleSize, in microns Prep Condition Initial 1 Week 0.25% SLS Ambient 0.3 μm0.8 μm with 25% API 5° C. 0.5 μm

2.3 Prototype Formulation Manufacturing

Given the comparable performance of both poloxamers as milling agents,poloxamer 407 was chosen for the prototype formulation instead ofpoloxamer 338 due to a more favorable toxicity profile. In order tofacilitate milling the quantities of test article required for study, atest batch was made at 30% API with a proportional increase in poloxamer407 to 2.4%. The median particle-size was successfully reduced to thesubmicron range with additional milling time:

TABLE 4 Particle-size reduction of concentrated suspension Milling TimeMedian Particle Size (min) (D50) 300 1.2 μm 360 0.8 μm 480 0.7 μm 5400.5 μm 600 0.1 μm

The concentrated suspension was diluted with deionized water to thetarget concentration of 10% API and that dilution was monitored forstability at ambient conditions. The particle size distribution wasstable for four weeks:

TABLE 5 Particle-size stability of diluted concentrate Time MedianInitial 0.1 μm 1 Week  0.2 μm 2 Weeks 0.2 μm 4 Weeks 0.2 μm

From these data, the prototype formulation for the1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazinenanosuspension was prepared, as follows:

Dissolve poloxamer 407 in approximately ⅓ of the purified water in asuitable milling container.

Suspend1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazinein the poloxamer 407 solution.

-   1. Add sufficient milling media to fill the container approximately    halfway.-   2. Roll container on roller mill until the D90 of the particle-size    distribution, measured by laser diffraction, is below a micron    (refractive index 1.637/i-value 0.1).-   3. Extract suspension from milling media.-   4. Rinse the media with a portion of the remaining water,    transferring the rinsate to the extracted suspension.-   5. Assay the suspension.-   6. Based upon the measured assay value, dilute the final suspension    with the remaining water to a concentration of 10%.-   7. Assay the suspension for confirmation.

More specifically, API was suspended at 300 mg/g in a 2.4% w/w solutionof poloxamer 407 in sterile water. 0.5 mm YTZ ceramic milling media(yttria-stabilized zirconia medium from Nikkato Corporation) was addedand the suspension was milled using a roller mill until the particlesize (D90), measured by laser diffraction, was not more than 2 micronand the median particle size (D50) was not more than 500 nm. Aftermilling, the suspension was diluted with sterile water to 100 mg/g API.

TABLE 6 Prototype Nanosuspension Formulation Component % w/w Water,Purified, USP 89.2% Poloxamer 407, NF 0.8%1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2- 10.0%methoxyquinoxalin-3-yl)aminocarbonyl]piperazine (API)

2.4 Prototype Manufacturing for GLP Studies

In detail, 472.9 grams of API was milled in three separate batches at30% concentration each. The milling media from each batch was extractedwith part of the remaining water and all the extracts were combined. Theassay value of the in-process suspension measured 201 mg/g (20.1%).Based on this value, the suspension was diluted to a final concentrationof 10% drug substance using sterile water for injection and anadditional poloxamer 407 to bring the total poloxamer concentration to2.05%. The net yield of the final suspension was 4,528 grams; at anassay value of 103 mg/g, the total API yield was 466.4 grams, or 98.6%of the original amount of API.

Example 3 Drug Product

The nanosuspension was pre-frozen using liquid nitrogen and lyophilizedin bulk on pre-chilled shelves to produce a dry powder. The dry powderwas manually weighed into a weighed funnel and then placed intohydroxypropyl methyl cellulose capsule shells (size 0) and packaged inquantities of 10 capsules per 30 ml high density polyethylene plasticbottle with a 5 g desiccant pack. Capsules containing 60 mg of activepharmaceutical ingredient were prepared. API lyophilized powder, 60 mgfill per capsule, has the following composition (Table 7) and batchformula (Table 8).

TABLE 7 Composition Amount Component per capsule API   50 mg Poloxamer407 10.25 mg

TABLE 8 Batch Formula Step Component Concentration Milling API 30.00%(w/w) Poloxamer 407  2.40% (w/w) Water 67.60% (w/w) Dilution and API10.00% (w/w) Poloxamer Poloxamer 407  2.05% (w/w) Addition Water 87.95%(w/w) Lyophilized API 82.99% (w/w) powder Poloxamer 407 17.01% (w/w)

Example 4 Stability of Drug Product

60 mg of Lyophilized powder API in size 0 hydroxypropyl methyl cellulosecapsule (to deliver 50 mg of API) stored in amber bottle with desiccantpacket and induction sealed. The capsules were stored at 5° C. or 25°C./60% relative humidity (RH) and the API and water content in capsule,particle size of API were analyzed. The data is shown in Table 9.

TABLE 9 Stability of drug product. Storage Test Results conditionParameter Initial 1 Month 2 Month 3 Month 6 Month 5° C. PSD (D10) 0.08μm (D10) 0.08 μm (D10) 0.08 μm (D10) 0.08 μm (D10) 0.08 μm (D50) 0.17 μm(D50) 0.19 μm (D50) 0.20 μm (D50) 0.19 μm (D50) 0.18 μm (D90) 1.6 μm(D90) 2.3 μm (D90) 2.1 μm (D90) 2.3 μm (D90) 1.7 μm Water  0.1% <0.1%<0.1% <0.1% <0.1% Content Assay 98.0% 98.9% 98.5% 100.9% 97.8% 25° C./PSD (D10) 0.08 μm (D10) 0.09 μm (D10) 0.17 μm (D10) 0.17 μm (D10) 0.18μm 60% RH (D50) 0.17 μm (D50) 0.24 μm (D50) 1.5 μm (D50) 1.5 μm (D50)1.6 μm (D90) 1.6 μm (D90) 2.8 μm (D90) 6.8 μm (D90) 6.7 μm (D90) 7.1 μmWater  0.1% <0.1% <0.1% <0.1% <0.1% Content Assay 98.0% 98.8% 98.1%97.0% 95.4% PSD = particle size distribution

As shown in Table 9, storage of drug product at 25° C./60% RH for 6months resulted in the increase of particle size and decrease of APIcontent. Storage at 5° C. showed no increase in particle size and nodecrease in API content for 6 months. Given the stability data in Table9, 5° C. was chosen for the storage of drug product.

CONCLUSIONS FROM EXAMPLES

Roller milling was found to be a simple, effective means by which toreduce the particle size of API. When processed with poloxamers asmilling aids, even at concentrations as high as 30% API, no fusing,ripening, discoloration or other detrimental physical phenomena wereobserved. Sodium lauryl sulfate, while it was shown to reduce theparticle size at very low concentrations, caused ripening, possibly dueto a cosolvency effect with the API. The use of a concentratedsuspension with an in-process assay allowed for multiple batches to beprocessed, combined and diluted to the desired concentration for thetoxicity study. This made possible the processing of over 470 grams ofAPI with nearly 99% recovery using laboratory-scale apparatus.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. All examples presented are representative and non-limiting.The above-described embodiments of the invention may be modified orvaried, without departing from the invention, as appreciated by thoseskilled in the art in light of the above teachings. It is therefore tobe understood that, within the scope of the claims and theirequivalents, the invention may be practiced otherwise than asspecifically described.

1. A stable composition comprising: (a) nanoparticles of compound offormula (1),

or pharmaceutically acceptable salts thereof, wherein X and Y areindependently N or C—R⁷; for the combination of variables R¹ and R²: R¹is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or halogen and R² is F; or R¹ isF and R² is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or halogen; R³ is C₁-C₃alkyl; and R⁴, R⁵, R⁶ and R⁷ are independently H, C₁-C₆ alkoxy, C₁-C₆alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkylcarbonyl, cyano, nitro or halogen;and (b) at least one surface stabilizer, wherein the nanoparticles havean effective median particle size (D50) of less than about 1,000 nm. 2.The composition of claim 1, wherein the compound of formula 1 is1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine.3. The composition of claim 1, wherein the at least one surfacestabilizer comprises a block copolymer of polyethylene oxide andpolypropylene oxide.
 4. The composition of claim 1, wherein the at leastone surface stabilizer comprises a poloxamer selected from a groupconsisting of poloxamer 407 or poloxamer
 338. 5. The composition ofclaim 1, in the form of a liquid suspension or a dry powder.
 6. Thecomposition of claim 1, wherein the composition is stable after storagefor at least four weeks.
 7. The composition of claim 1, wherein theeffective median particle size is of less than about 500 nm.
 8. Thecomposition of claim 1, wherein the ratio (wt/wt) of the compound offormula (1) to surface stabilizer is from about 100:1 to about 5:1.
 9. Amethod of making a storage stable composition comprising preparing amixture of (a) particles of a compound of formula (1),

or pharmaceutically acceptable salts thereof, wherein X and Y areindependently N or C—R⁷; for the combination of variables R¹ and R²: R¹is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or halogen and R² is F; or R¹ isF and R² is hydrogen, C₁-C₃ alkoxy, C₁-C₃ alkyl or halogen; R³ is C₁-C₃alkyl; and R⁴, R⁵, R⁶ and R⁷ are independently H, C₁-C₆ alkoxy, C₁-C₆alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkylcarbonyl, cyano, nitro or halogen;and (b) at least one surface stabilizer, and reducing the size of theparticles of a compound of the formula (1) under conditions sufficientto provide a nanoparticulate suspension having an effective medianparticle size of less than about 1,000 nm, the composition is stable.10. The method of claim 9, further comprising drying the nanoparticulatesuspension, for example by lyophilization, to form a powder.
 11. Themethod of claim 9, wherein the mixture comprises from about 5% to about50% of the compound of formula 1 and from about 0.1% to about 5% of thesurface stabilizer.
 12. The method of claim 9, wherein the powdercomprises from about 75% to about 90% of the compound formula 1 and fromabout 10% to about 25% of the surface stabilizer.
 13. The method ofclaim 9, wherein the ratio of the compound of formula (1) to surfacestabilizer is from about 100:1 to about 5:1.
 14. The method of claim 9,wherein the reducing comprises grinding, homogenizing or precipitating.15. The method of claim 14, wherein the reducing comprises grinding forfrom about 360 to about 600 minutes.
 16. The method of claim 9, whereinthe compound of formula 1 is1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl]piperazine.17. The method of claim 9, further comprising adding water to themixture after reducing the size of the particles.
 18. The method ofclaim 9, further comprising adding a poloxamer solution to the mixtureafter reducing the size of the particles.
 19. A pharmaceuticalcomposition comprising the composition of claim 1 and at least onepharmaceutically acceptable excipient.
 20. The pharmaceuticalcomposition of claim 19, wherein the at least one pharmaceuticallyacceptable excipient comprises water or hydroxypropyl methylcellulose.21. The pharmaceutical composition of claim 19, wherein the compositionis an oral dosage form or a parenteral dosage form.
 22. Thepharmaceutical composition of claim 19, comprising about 0.01 to about250 mg of the compound of formula (1).
 23. The method of claim 1,wherein said tumor is selected from the group consisting of: ovarytumors, breast tumors, prostate tumors, liver tumors, lung tumors,kidney tumors, colon tumors, pancreatic tumors, and stomach tumors.